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O-GlcNAcylation is a dynamic and reversible post-translational modification of cytonuclear molecules that regulates cellular signaling. Elevated O-GlcNAcylation is a general property of cancer and plays a critical role in cancer progression. We previously showed that the expression of FOXM1, a critical oncogenic transcription factor widely overexpressed in solid tumors, was elevated in MKN45 cells, a human gastric cancer cell line, by the O-GlcNAcase inhibitor Thiamet G (TMG), which induces augmented O-GlcNAcylation. Here, we identified FBXL2 E3 ubiquitin ligase as a new target of O-GlcNAcylation. Consistent with the results in MKN45 cells, FOXM1 expression was increased, accompanied by its decreased ubiquitination and degradation by TMG in the other gastric cancer cell lines, including NUGC-3 cells. We found that FBXL2 ubiquitinated FOXM1, and the interaction with FBXL2 and ubiquitination of FOXM1 were reduced by TMG in NUGC-3 cells. Interestingly, FBXL2 was also ubiquitinated, which was promoted by TMG in the cells. Moreover, FOXM1 expression and cell proliferation were reduced in FBXL2-induced NUGC-3 cells, and the reductions were attenuated by TMG, indicating that FOXM1 was stabilized by O-GlcNAcylation-mediated degradation of FBXL2 to induce cancer progression. These data suggest that elevated O-GlcNAcylation contributes to cancer progression by suppressing FBXL2-mediated degradation of FOXM1., (Copyright © 2019 Elsevier Inc. All rights reserved.)

O-GlcNAcylation is a dynamic post-translational modification of cytonuclear proteins for intracellular signaling. Elevated O-GlcNAcylation is a general feature of cancer and contributes to cancer progression, and recent studies indicate the contribution to increasing incidence of various types of cancer in diabetic patients. However, the role of O-GlcNAcylation in tumor progression is not fully elucidated. Forkhead box M1 (FOXM1), a master mitotic transcription factor, has been implicated in all major hallmarks of cancer, and is wildly expressed in solid tumors. Given that FOXM1 expression was reported to be elevated in gastric cancer, we examined the effect of high glucose or an inhibitor of O-GlcNAc hydrolase, Thiamet G (TMG), on FOXM1 protein expression in a human gastric cancer cell line, MKN45 cells, and confirmed that FOXM1 protein level and the cell proliferation were upregulated. To investigate the molecular mechanisms by which FOXM1 protein expression is regulated by O-GlcNAcylation, the effect of high glucose and TMG on FOXM1 ubiquitination was examined in MKN45 cells. As a result, the ubiquitination and degradation of FOXM1 protein were both suppressed by high glucose and TMG treatment. However, the O-GlcNAcylation was not detected on FOXM1 but not on GSK-3β. High glucose and TMG treatment increased phospho-serine 9 GSK-3β, an inactive form, and the degradation of FOXM1 protein was suppressed by treatment of GSK-3β inhibitors in MKN45 cells. Taken together, we suggest that high glucose and elevated O-GlcNAcylation stabilize FOXM1 protein by its reduced degradation via GSK-3β inactivation in MKN45 cells, suggesting that the higher risk of gastric cancer in diabetic patients could be partially due to O-GlcNAcylation-mediated FOXM1 stabilization., (Copyright © 2017 Elsevier Inc. All rights reserved.)

W9 is a peptide that abrogates osteoclast differentiation via blockade of nuclear factor-κB ligand (RANKL)-RANK signaling, which activates bone formation. However, W9 stimulated osteogenesis in osteoblasts and mesenchymal stem cells. The present study demonstrated that the W9 peptide promoted osteogenic differentiation of human adipose-derived stem cells (hAdSCs) even under non-osteogenic differentiation culture conditions. W9-treated hAdSCs exhibited several osteocalcin-expressing cells and great mineralization compared to the BMP2-treated hAdSCs, which suggests that the W9 peptide had potent osteogenic potential in hAdSCs. W9 treatment also markedly enhanced the phosphorylation of p38, JNK, Erk1/2, and Akt, and BMP2 treatment only enhanced the phosphorylation of p38 and Erk1/2 in hAdSCs. hAdSCs did not express the RANKL gene, but W9 treatment upregulated Runx2, Collagen type 1A1 and TGF receptor genes and increased Akt phosphorylation. These results suggest that the W9-induced potent osteogenic induction was attributed to activation of TGF and the PI3 kinase/Akt signaling pathway in hAdSCs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Phospholamban (PLN) regulates cardiac type sarco (endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) via Ser(16)-phosphorylation. During heart failure, PLN expression is downregulated with SERCA2a; however, the mechanism of its regulation is not fully understood. Phosphorylation triggers protein degradation and because PLN phosphorylation is upregulated in failing hearts, we examined whether PLN is degraded by Ser(16)-phosphorylation. Cells overexpressing PLN exhibited its degradation post isoproterenol (Iso), forskolin, or 3-isobutyl-1-methylxanthine (IBMX) addition. Moreover, this degradation was inhibited by a cAMP-dependent protein kinase (PKA) inhibitor--H89. Co-immunoprecipitation revealed that Lys(3) of PLN was oligo-ubiquitinated when ubiquitin was overexpressed, and was degraded by Iso treatment. However, when co-expressed with SERCA2a, oligo-ubiquitinated PLN at Lys(3) was not degraded by Iso treatment. In failing hearts from 16 week-old TgPLN(R9C) mice, oligo-ubiquitinated PLN levels increased and PLN expression was downregulated. Furthermore, SERCA2a mRNA levels in TgPLN(R9C) mice hearts were lower than that in wild type mice; however, PLN mRNA levels showed no changes. In another heart failure model, MG132 treatment reversed PLN degradation. These data suggest that PLN is, at least partially, oligo-ubiquitinated at Lys(3) and degraded through Ser(16)-phosphorylation-mediated poly-ubiquitination during heart failure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Non-steroidal anti-inflammatory drug (NSAID)-induced epithelial cell damage occurs not only in the stomach but also in the intestines and colon. Although several studies have investigated the related mechanism underlying lower gastrointestinal tract injury, the details of this mechanism are still unclear. Since it was reported that protein degradation might play an important role, herein, we focused on one of the major ubiquitin E3 ligases, the von Hippel-Lindau protein (pVHL). To understand whether pVHL is involved in the observed cell damage, we examined whether indomethacin (IM) treatment affects pVHL expression in the rat IEC6 intestinal epithelial cell line. We showed that pVHL was upregulated after IM treatment through increased oxidative stress. pVHL siRNA prevented cell injury after IM treatment. Furthermore, the collagen I and HIF-1α protein band intensities were both decreased after IM treatment, whereas MG132 reversed the proteins' downregulation, indicating that the IM treatment-induced downregulation was due to the degradation through pVHL-mediated polyubiquitination. Co-immunoprecipitation showed that pVHL interacted with both collagen I and HIF-1α. The degradation of collagen I and HIF-1α after IM treatment was reversed by siVHL or a Mn-SOD mimetic, Mn(III)TMPyP. The expression of collagen I and HIF-1α was correlated with pVHL expression level, whereas only HIF-1α, not collagen I, was upregulated after the treatment of a prolyl hydroxylase inhibitor, CoCl2. The effect of pVHL on the intestinal epithelium after IM treatment was also tested in vivo. Western blot analyses were used to test whether pVHL's protein expression level might increase after oral administration of IM to mice, and which showed that IM upregulated pVHL expression and degraded collagen I and HIF-1α, consistent with the data obtained in IEC6 cells. These data suggested that intestinal epithelial cells were injured after IM treatment through the pVHL overexpression-induced degradation of collagen I or HIF-1α. Therefore, pVHL might be a molecular target for IM-induced intestinal epithelial cell injury., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A structurally unique isoxazoline class compound, A1443, exhibits antiparasitic activity against cat fleas and dog ticks comparable to that of the commercial ectoparasiticide fipronil. This isoxazoline compound inhibits specific binding of the gamma-aminobutyric acid (GABA) receptor channel blocker [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) to housefly-head membranes, with an IC(50) value of 455pM. In contrast, the IC(50) value in rat-brain membranes is>10muM. To study the mode of action of this isoxazoline, we utilized MdGBCl and MdGluCl cDNAs, which encode the subunits of housefly GABA- and glutamate-gated chloride channels, respectively. Two-electrode voltage clamp electrophysiology was used to confirm that A1443 blocks GABA- and glutamate-induced chloride currents in Xenopus oocytes expressing MdGBCl or MdGluCl channels, with IC(50) values of 5.32 and 79.9 nM, respectively. Blockade by A1443 was observed in A2'S-MdGBCl and S2'A-MdGluCl mutant channels at levels similar to those of the respective wild-types, and houseflies expressing A2'S-MdGBCl channels were as susceptible to A1443 as standard houseflies. These findings indicate that A1443 is a novel and specific blocker of insect ligand-gated chloride channels., (Copyright 2009 Elsevier Inc. All rights reserved.)

Fucosylation is one of the most important oligosaccharide modifications and is involved in cancer and inflammation. Recently, fucosylated haptoglobin was identified as a possible tumor marker for pancreatic cancer. The molecular mechanism underlying increases in fucosylated haptoglobin in sera of patients with pancreatic cancer seems to be complicated. Our previous study [N. Okuyama, Y. Ide, M. Nakano, T. Nakagawa, K. Yamanaka, K. Moriwaki, K. Murata, H. Ohigashi, S. Yokoyama, H. Eguchi, O. Ishikawa, T. Ito, M. Kato, A. Kasahara, S. Kawano, J. Gu, N. Taniguchi, E. Miyoshi, Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation, Int. J. Cancer 118 (11) (2006) 2803-2808] demonstrated that pancreatic cancer cells secrete a factor, which induces the production of haptoglobin in hepatoma cells. In the present study, we found that interleukin 6 (IL6) expressed in pancreatic cancer is a factor that induces the haptoglobin production, using a neutralizing antibody for IL6. Real-time PCR analyses revealed the up-regulation of fucosylation regulatory genes after IL6 treatment, resulting increases in fucosylated haptoglobin being revealed by a lectin ELISA. This pathway could be one of the possible mechanisms underlying increases in haptoglobin in sera of patients with pancreatic cancer.

A robust cross-link between Gln(23) in phospholamban (PLN) and Lys(328) in the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) is formed in the presence or absence of oxidant and is susceptible to both PLN phosphorylation and SERCA1a Ca(2+) binding. This cross-link provides precisely the evidence needed to support our earlier proposal that collision of the PLN transmembrane helix at Asn(27) with the cytosolic extension of M4 at Leu(321) leads to unwinding of the helix. In a study of site-specific interactions among PLN, sarcolipin (SLN), and SERCA1a, we determined that mutations of some specific amino acids in PLN or SLN diminish either the super-inhibition imposed on SERCA1a function by the PLN-SLN binary complex or the physical interactions between PLN and SLN or both. These results have led to a revision of our earlier model for the PLN-SLN-SERCA1a complex.

Heparin-binding EGF-like growth factor (HB-EGF), which is a potent mitogen for vascular smooth muscle cells (SMC) and fibroblasts, has been reported to be strongly implicated in atherosclerosis and wound healing. HB-EGF mRNA is known to be induced by thrombin, angiotensin-II, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and HB-EGF itself in SMC. In vascular endothelial cells (EC), its mRNA is induced by tumor necrosis factor-alpha and interleukin-1beta. Only phorbol 12-myristate 13-acetate is a common inducer for HB-EGF mRNA. The present study shows that calcium ionophore A23187 also induced HB-EGF mRNA in both SMC and in EC and that both intracellular reactive oxygen species (ROS) and an increase in calcium levels were essential for the induction of this growth factor mRNA. While HB-EGF caused an increase in both intracellular ROS and calcium in SMC, it increased only calcium, but not the intracellular ROS in EC. When the intracellular ROS was elevated by treatment with hydrogen peroxide (H2O2) or by depletion of glutathione by buthionine sulfoxamine, both HB-EGF and thrombin were observed to upregulate HB-EGF mRNA in EC. These data suggest that H2O2, produced by activated leukocytes in inflammatory lesions, upregulates HB-EGF mRNA by cooperating with thrombin, angiotensin-II, and the above growth factors. Since activated macrophages under the EC are thought to elevate the ROS in neighboring EC, this mechanism might play a major role in the progression of atherosclerosis and for wound healing., (Copyright 1999 Academic Press.)

Here we investigated the involvement of the non-receptor protein-tyrosine kinase p72syk in formyl methionyl-leucyl-phenylalanine (fMLP) receptor signaling. The activity of p72syk began to rise from 15 s and reached to maximum within 2-5 min after 5 microM fMLP stimulation in porcine polymorphonuclear neutrophils (PMNs). Cyclic AMP (cAMP)-elevating agents, prostaglandin E2 (PGE2) and forskolin, or dibutyryl cAMP partially suppressed p72syk activities stimulated by fMLP in PMNs. Pretreatment with an inhibitor of cAMP-dependent protein kinase abolished the suppression of the fMLP-induced p72syk activation by these cAMP-elevating agents. It was also observed that cAMP-dependent protein kinase phosphorylates p72syk on serine residues in vitro. These results indicate a possibility that cAMP-dependent protein kinase negatively regulates the activation of p72syk in fMLP-receptor signaling.

We previously reported a molecular cloning of porcine gene syk encoding a non-receptor type 72-kDa protein-tyrosine kinase (Taniguchi et al. (1991) J. Biol. Chem. 266, 15790-15796). In this study, we have demonstrated that p72syk is expressed in porcine platelets at 0.1-0.2% of total protein and that the lectin wheat germ agglutinin induces an activation of p72syk against both auto- and exogenous-substrate-phosphorylation in porcine platelets. The activation of p72syk was abrogated by the coexistence of N-acetyl-D-glucosamine with wheat germ agglutinin. These data suggest that p72syk is a candidate of responsible protein-tyrosine kinase for platelet activation and that cell surface glycoprotein is involved in the activation of p72syk in platelets.

Effect of membrane phospholipids on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) has been studied. Using poly(Glu Na, Tyr)4:1 as a substrate, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine had stimulatory effects on that phosphorylation activity, however phosphatidic acid had inhibitory and phosphatidylinositol had no effects. Similar results were obtained using[Val5]angiotensin II as a substrate. On the other hand using basic protein (H2B histone and myelin basic protein) as substrates, phosphatidic acid stimulated the activity of CPTK-40, while phosphatidylinositol inhibited the activity. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine caused different effect on the activity of CPTK-40 depending on the substrate employed. However using acidic protein (tubulin and casein) as substrates, the activity of CPTK-40 was neither stimulated nor inhibited by any phospholipids. These results suggest that phospholipids may modulate the activity of CPTK-40.

Cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) is strongly activated by poly-L-lysine using bovine serum albumin, ovalbumin, phosphorylase b, calmodulin and H1 histone as substrate proteins. However, this polyamine inhibited the enzyme activities when myelin basic protein, tubulin and H2B histone were used as substrate proteins. These stimulatory and inhibitory effects on CPTK-40 are not specific for polylysine, but polyarginine and polyornithine have similar effects on this phosphorylation reaction. Effect of poly-basic amino acids on CPTK-40 seems to be mainly on the substrate proteins, rather than on the enzyme itself.